Influenza virus activation of the interferon system

Abstract

The host interferon (IFN) response represents one of the first barriers that influenza viruses must surmount in order to establish an infection. Many advances have been made in recent years in understanding the interactions between influenza viruses and the interferon system. In this review, we summarise recent work regarding activation of the type I IFN response by influenza viruses, including attempts to identify the viral RNA responsible for IFN induction, the stage of the virus life cycle at which it is generated and the role of defective viruses in this process.

Keywords: Influenza viruses; Innate immunity; Interferon; RIG-I.

Fig. 1

Heterogeneity in activation of the IFN induction cascade in cells infected with influenza viruses. A549 cells expressing GFP under the control of an IFN-β promoter (A549/pr(IFN-β).GFP; Chen et al., 2010) were infected with 5 PFU/cell of PR8, Udorn or WSN strains of influenza A virus. Cells were left uninfected or infected with 5 PFU/cell Sendai virus (Cantell strain) as negative and positive controls for GFP expression respectively. At 16 h post-infection, monolayers fixed and stained with antibody raised against disrupted X31 virus and DAPI; GFP (green), viral protein expression (red) and cell nuclei (blue) were subsequently examined by confocal microscopy.

Fig. 2

The panhandle and corkscrew structures of the influenza virus promoter. The panhandle (A) and corkscrew (B) conformations of influenza virus vRNA and the corkscrew conformation of cRNA (C) are shown. Sequences shown from segment 5 (NP) of the WSN strain of influenza virus.

Fig. 3

Activation of the IFN induction cascade by influenza virus is sensitive to actinomycin D but not cycloheximide. A549 cells were infected with 5 PFU/cell of an NS1-defective Udorn virus (Ud-Δ99; Jackson et al., 2010) or a DI-rich preparation of PIV5 (PIV5-VΔC vM2; Killip et al., 2011), in the presence of 1 μg/ml actinomycin D (ActD) or 50 μg/ml cycloheximide (CHX) or were left untreated (UT). At 16 h post-infection, cell lysates were prepared and probed for phospho-IRF3 (p-IRF3), total IRF3, viral proteins and actin.

Fig. 4

DIs in influenza virus preparations correlate with an enhanced ability to activate the interferon system. DI-preparations of PR8 were obtained by four sequential high multiplicity passages of the vM0 virus through MDCK cells. The total number of virus particles (haemagglutination units; HAU) and the plaque-forming titre (plaque-forming units; PFU) were obtained by haemagglutination assay and plaque assay respectively. A549 cells expressing GFP under the control of an IFN-β promoter (A549/pr(IFN-β).GFP; Chen et al., 2010) were infected with the low DI (vM0) or DI-rich (vM4) preparations of PR8 at equivalent infectious titre (0.3 PFU/cell) or an equivalent number of total virus particles (1 × 10⁴ HAU/cell). At 16 h post-infection, cells were trypsinised, fixed and analysed by flow cytometry for GFP expression (A). Duplicate monolayers were harvested for immunoblotting (B); lysates were probed for markers of activation of the interferon system (phospho-IRF3, GFP and MxA), viral NP expression and actin (as a loading control). The IFN present in culture media was estimated by CPE-reduction bio-assay, and the relative units of IFN (RUI) for each condition are indicated beneath the immunoblot panels.